(A) Study pathogenesis and pathophysiology of high biocontainment viral pathogens utilizing molecular technologies including reverse genetics systems: We have developed a mouse model of CCHF in which infected mice develop severe disease but ultimately most recover. Although mice developed an inflammatory immune response along with severe liver and spleen pathology, these mice also developed CCHFV-specific B and T-cell responses and were protected from re-infection. This model provides a valuable tool to investigate how host immune responses control acute CCHFV infection, how these responses may contribute to the severe disease seen in CCHFV-infected humans and develop therapeutic interventions that promote protective immune responses. (Hawman et al., J Virol 2019) We developed the first CCHFV nonhuman primate disease model. Cynomolgus macaques exhibited hallmark signs of human CCHF with remarkably similar viral dissemination, organ pathology and disease progression. Histopathology showed infection of hepatocytes, endothelial cells and monocytes and fatal outcome seemed associated with endothelial dysfunction manifesting in a clinical shock syndrome with coagulopathy. This non-human primate model will be an invaluable asset for CCHFV countermeasures development. This year we performed a serial sacrifice study to determine the disease progression using virological, immunological and histopathological techniques. (Haddock et al. Nature Microbiol 2018; Haddock et al., unpublished data) The mouse model for Ebola virus (EBOV) is a well-established small animal disease model. However, recently multiple studies reported surviving control animals when evaluating treatment or vaccine approaches. Therefore, we analyzed the severity of disease and lethality of mouse-adapted (MA-) EBOV infection in six different mouse strains. We identified outbred CD-1 mice to be the only strain tested resulting in uniform lethality when infected with different doses of MA-EBOV or reverse genetics-generated MA-EBOV. This information will be critical for future countermeasure development. (Haddock et al., J Infect Dis 2018) We compared the pathogenesis of Ravn virus (RAVV) and Marburg virus (MARV) strains Angola, Musoke, and Ozolin in rhesus and cynomolgus macaques. Our results reveal the most pathogenic MARV strain to be Angola followed by Musoke, whereas Ozolin is the least pathogenic. We also demonstrate that RAVV is highly pathogenic in cynomolgus macaques, but less pathogenic in rhesus macaques. This information expands our knowledge on pathogenicity and virulence of marburgviruses which will be critical for countermeasure development. (Nicholas et al., J Infect Dis 2018) We have developed a disease model for Reston ebolavirus (RESTV) in a commercial pig breed. Young pigs, ranging in age from 3 7 months, were highly susceptible to oral-nasal inoculation of RESTV. The animals developed acute severe respiratory distress with high but not uniform lethality. Most Young pigs succumb within a week of infection; some animals recover from severe disease. RESTV replicates mainly in respiratory tissues and virus is shed through mucosal membranes of the oronasal tract. This is the first RESTV disease model as previous work in pigs has resulted in asymptomatic infection. The model will be instrumental for countermeasure development against a potential livestock pathogen. (Haddock et al., in preparation) (B) Study immune responses to infection and vaccination of high containment viral pathogens and develop new vaccine candidates: The VSV vaccine efforts are reported under the 'Trivalent Filovirus Vaccine' project. We have developed a safe and efficacious formulation of insect-cell derived adjuvanted protein vaccines for Ebola virus (EBOV). Vaccine candidates containing the EBOV glycoprotein with or without matrix proteins VP24 and VP40 formulated with one of three different adjuvants were tested in guinea pigs for immunogenicity and efficacy against lethal EBOV challenge. The results demonstrated that these vaccine candidates engendered high titers of antigen-specific antibodies in immunized animals and two of these vaccine candidates afforded complete or nearly complete protection against lethal challenge. As this vaccine has a good safety profile, it will be further investigated. (Lehrer et al., Vaccine 2019) We report the development and assessment of the protective efficacy of an adenovirus (Ad)-based CCHFV vaccine expressing the nucleocapsid protein (N) of CCHFV (Ad-N) in a lethal immunocompromised mouse model of CCHF. The results show that Ad-N can protect mice from CCHF mortality and that this platform should be considered for future CCHFV vaccine strategies. Further vaccine efforts based on alphavirus replicons expressing CCHFV N and glycoprotein Gn and Gc or all combined are under design and evaluation. (Zivcec et al., PLoS Negl Trop Dis 2018) (C) Study vector/reservoir transmission of high containment viral pathogens using appropriate animal models: We continued to study infection kinetics of Lassa virus in the Mastomys reservoir utilizing a unique colony established here at RML. The animals support virus replication and shedding for several weeks before Lassa virus gets cleared. The model will allow for important transmission studies. We also developed immunological tools to study host responses in Mastomys. A wildlife vaccine project has been designed and started with the aim to stop transmission among the reservoir species and to humans. (Huau-Tang et al., manuscript in preparation) (D) Utilize in vitro and in vivo systems to study the interactions between viral pathogen or viral components and host cells and develop new antiviral strategies: We utilized a mouse model to evaluate and compare efficacy of ribavirin and favipiravir against lethal CCHFV challenge (strains Hoti and 10200). While ribavirin treatment suppressed viral loads, it was unable to prevent development of terminal disease. In contrast, favipiravir showed clinical benefit even when administered late in the clinical progression of CCHF. More recently, we have tested favipiravir in the CCHFV Cynomolgus macaque disease model. In this model, the benefit of favipiravir showed only limited benefit. Nevertheless, given the bad performance of ribavirin in animal models, we propose to start human trials with favipiravir as the drug seems more potent against CCHFV. (Hawman et al., Antiviral Res 2018; Hawman et al., unpublished) We have tested polyclonal and monoclonal antibodies for efficacy against filovirus infections in animal models. First, we utilized transchromosomal bovines (TcB) vaccinated with a DNA plasmid encoding Ebola virus glycoprotein sequence to produce human polyclonal antibodies. When administered 1-day post infection, these TcB polyclonal antibodies provided partial protection and resulted in a 50% survival rate following a lethal challenge of Ebola virus in rhesus macaques. Next, we attempted to improve the therapeutic efficacy of the neutralizing mAb M4 against Marburg virus challenge by combining treatment with 1 or 2 of blocking but non-neutralizing mAbs 126-15 and 127-8. We found that single-dose treatment early after infection with the neutralizing mAb M4 or any of the mAb combinations resulted in similar protection in the hamster model. However, a single-dose treatment with the cocktail of all 3 mAbs provided the best protection in delayed treatment, with 67%-100% of the animals surviving a lethal challenge depending on the time of treatment. (Rosenke et al., J Infect Dis 2018; Marzi et al., J Infect Dis 2018) (E) Study the epidemiology and ecology of high biocontainment pathogens utilizing newly developed rapid, sensitive and specific diagnostic test systems including those that can be applied under field conditions: Please see Annual Report on Mali ICER project.